RUFUS Requirements

RUFUS requires the use of the gcc-4.9.2 compiler. Please make sure that are using the gcc-4.9.2 compiler before you proceeed. If you are not using gcc-4.9.2 as your default compiler, or if CMake cannot detect the location of your gcc compiler, RUFUS will not build and install properly.

RUFUS requires Samtools. Please make sure that samtools is globally installed in your environment. RUFUS will not run without a working samtools.

Testing RUFUS

To make sure that RUFUS was successfully built, we provide users with a test run script to run RUFUS on a small test set of data with a small test reference, and default parameters. To test RUFUS, simply run

cd testRun
bash runTest.sh

NOTE: Make sure that runTest.sh is called directly from the testRun directory, or the testRun script will not be able to find the appropriate resources.

All data for this run is contained in the resources dir, and nothing needs to be provided by the user to test RUFUS.

At the end of a successfull test run, you should see a file named

testRun/Child.bam.generator.V2.overlap.hashcount.fastq.bam.vcf

This file should contain a single varient call. The call should look exactly as follows:

If you were unable to reproduce this call, something went wront with the RUFUS install, and you should get the test run to work before proceeding further. If you are unable to reproduce a successfull test run, please contact me at JAndrewRFarrell@gmail.com

Running

RUFUS is primarily used to find mutations unique to a proband sample, that are not found in the control samples

-s,--subject: bam file containing the subject of interest (REQUIRED)
-c, --controls: bam files containing the control subjects (REQUIRED)
-t,--threads: number of threads to use (REQUIRED) (min 3)
-k,--kmersize: size of Kmer to use (REQUIRED)
-r,--ref: file path to the desired reference file to create VCF (REQUIRED)
-m,--min: overwrites the minimum k-mer count to call variant (OPTIONAL, Do not provide a min unless you are sure what you want)
-h,--help: HELP!!!!!!!!!!!!!!!

We recommend a kmer size of 25, 40 threads, and to NOT provide RUFUS with the optional --min parameter

Providing a reference file.

After RUFUS has identified reads containing mutant kmers, the reads must be aligned to a reference fasta file. Any fasta file can be used as a reference, as long as the fasta file has been indexed for BWA. If a fasta has been indexed by bwa, there will be reference files with the following extensions: pac, .ann, .abm, .bwt, sa. In order to prepare a reference fasta for bwa, simply type:

bwa index -a bwtsw reference.fa
samtools faidx reference.fa

This will produce the BWA index files, and the fasta file index respectively. Make sure that the bwa index files and the fasta index file are in the same directory as reference.fa

Ubuntu dependencies

In order for RUFUS to run on a fresh Ubuntu build, all of the following packages must be installed: